Activation of cAMP-dependent protein kinase (PKA) represents the major mechanism by which cell surface receptors coupled to adenylate cyclase are able to regulate cellular functions. The phosphorylation of cellular substrate proteins such as transcription factors, ion channels, and cytoskeletal proteins by the catalytic (C) subunit of PKA triggers changes in cellular gene transcription, excitability, and morphology,. During the previous grant period we characterized PKA regulation of protein secretion in AtT-20 cells and isolated cDNAs for an inhibitor protein of PKA. The protein kinase inhibitors (PKIs) are small proteins able to potently and specifically interact with the catalytic (C) subunit of PKA to inhibit the phosphorylation of cellular proteins. These PKI proteins are evolutionarily conserved but their exact role in regulated cellular responses to elevation of cAMP is not understood. Recent work in the area has supported a physiological role for the PKI proteins. The research proposed for the new grant period will combine biochemical, molecular biological, immunological and cell biological techniques to elucidate the role of PKIs in regulating cellular responses to PKA activation. Specifically, cDNA and genomic clones will be isolated for the known PKI isoforms as well as for novel PKI isoforms. These cloned DNAs will be used to quantitate the level of PKI mRNA in various tissues and cell lines by Northern blot analysis and in situ hybridization. The DNAs will also be used to generate antibodies to the specific PKI isoforms for quantitation of the PKI proteins during development, after hormonal stimulation, and during the cell cycle. Transfection of DNAs coding for the PKI isoforms and C subunit will be used to identify C-PKI complexes formed in vivo and to identify specific mutants of both PKI and C subunit which alter C-PKI interactions. These mutants along with the normal PKI proteins will be used to define effect of PKI subunit expression on C subunit stability and activity. Finally, the effects of C-PKI interactions on specific cellular responses such as gene transcription and protein secretion will be determined. The result of these investigations will be a detailed understanding of the role of PKI proteins in modulating the cellular response to activation of the PKA signal transduction pathway. The long-range goal of this work is to understand all of the components of the PKA signal transduction system and their mechanisms of control of key cellular processes such as protein secretion.